This application proposes to study, specifically, the structure and function of the bile salt stimulated cholesterol esterase. This protein is made primarily in the pancreas and plays a critical role in the intestinal absorption of dietary cholesterol. Part I of this research will complete the cloning and sequencing of the human cholesterol esterase cDNA previously isolated by screening of a human mammary tumor (T-47D) cDNA library with a rat pancreatic cholesterol esterase cDNA. Comparing the sequences of the rat and human proteins with other serine esterases will provide significant information on the domain structure of the cholesterol esterase. The second aim of the proposal is to identify the specific domains of cholesterol esterase by chemical modification techniques. Preliminary data have implicated the involvement of a serine, a histidine, and a carboxylic acid in the active site domain of cholesterol esterase. The specific residues involved with catalysis will be identified by modification with [3H]DFP, [14C]p-bromophenacyl bromide, and carbodiimide/[14C]glycine ethyl ester, respectively. Specific amino acids labeled with these reagents in the absence, but not in the presence, of substrate protection will be identified by peptide sequencing methods. Additionally, the tyrosine residue important for bile salt binding and activation will be determined by modification with trinitromethane in the presence or absence of bile salt. Once the key residues for each functional domains have been identified, the third aim of the study will use site-specific mutagenesis technique to alter specific sequences within these domains and to express both normal and mutant proteins in CHO cells for characterization. The fourth objective of the research is to isolate and sequence the human cholesterol esterase gene. The intron-exon junction and the 5' and 3' DNA regions will be determined. Possible regulatory elements controlling the expression of the cholesterol esterase gene will be identified. In particularly, cis-acting elements that confer tissue- specific expression and the expression of the cholesterol esterase in mammary gland during lactation will be determined. Chimeric genes will be constructed using DNA from the 5' region of the gene as regulator for expression of a reporter gene. Mutagenesis will then be used to alter the regulatory elements and to determine key residues in this domain important for gene regulation.